Study Of Growth Changes In Oral Microorganisms Under Controlled Oxygen Conditions

The special physiological structure of the oral cavity provides different ecological niches for microbial adhesion and colonization,however,because the oral cavity is exposed to the external environment,the microbial community therein is highly susceptible to external factors such as oxygen content,temperature,and climatic factors.The stability of the oral microbial community plays a crucial role in maintaining oral health and disease susceptibility.As a precision microfluidic system that can simulate the human microenvironment in vitro,the organ chip provides a good in vitro experimental platform for in vivo microbiome-related research.Currently,a large number of studies have used organochip to reveal the pathogenic mechanisms that exist between the human microbiome and various diseases in vivo.However,there are fewer studies related to the study of oral microbiology using oral microarrays,and at the same time,there is a relative lack of studies on the relationship between oral microbiology and environmental factors.Objective: In this project,a fluidic device was prepared firstly that can simulate in vitro oxygen concentration and saliva flushing at different altitudes.The device used PDMS to achieve an anaerobic environment inside the device,and the circulation channel formed by the peristaltic pump carried oxygen to form a relatively high oxygen region at the bottom of the upper chamber,so that a gradual oxygen gradient was formed in the agar block from bottom to top to achieve controlled oxygen distribution.The solid agar block is also used to cultivate microorganisms,which bionically simulate the three-dimensional growth structure of oral microorganisms in biofilm.To equip the device with in vitro simulation of oxygen conditions at different altitudes as well as an oral fluid environment for the simulation of microbial culture.Secondly,the growth state and biofilm formation ability of oral microorganisms under different oxygen gradients were investigated in combination with the traditional laboratory culture mode,and the influencing mechanisms causing the changes of microbial growth state and biofilm formation ability were investigated to verify the accuracy of the in vitro device results using the laboratory experimental results.Finally,16 S r RNA high-throughput sequencing was used to explore the real changes in oral microbial community structure in populations living at different altitudes and the length of time living at high altitudes,and to analyze the differences between oral microbial gene functions.The results of oral microbial changes under real altitude changes verified the accuracy of the device simulation results and laboratory simulation results.This project proposes an in vitro microbial culture device that incorporates external environmental changes,providing a new platform and new ideas for the study of oral microorganisms in high-altitude populations,with a view to providing a corresponding theoretical basis for the prevention and treatment of oral diseases in high-altitude populations.Methods:(1)A fluidic device for human oral microbial growth environment that can simulate in vitro oxygen concentration at different altitudes and with salivary flushing is prepared based on the currently existing anaerobic model of human organs.The oxygen gradient inside the device is detected by an oxygen probe.(2)In vitro simulation of planktonic growth and biofilm formation of oral microorganisms under different altitude oxygen conditions using the device and conventional laboratory methods.(3)Preliminary investigation of biofilm formation mechanisms using common oral pathogenic bacteria Streptococcus pyogenes as an example.(4)The 113 supragingival plaques of 71 study subjects from different altitudes were collected and subjected to 16 S r RNA high-throughput sequencing to investigate the changes in the constitutive structure of the oral microorganisms and their diversity at different altitudes in authentic environments,and the sequencing results were compared with the device simulation results to verify the accuracy of the in vitro simulation device.Results:(1)An oxygen gradient was formed in the in vitro simulator from bottom to top with gradually decreasing oxygen content.(2)The microorganisms cultured in the device were consistent with the results of conventional laboratory culture,showing that Streptococcus pyogenes showed higher biofilm formation ability and growth status in the high-altitude group.(3)The growth and biofilm-forming ability of S.mutans were significantly inhibited by the addition of 50 μg/m L of D-cycloserine(DCS);meanwhile,the addition of exogenous D-alanine(D-Ala)compensated the antibacterial effect of DCS on S.mutans.(4)The genus Streptococcus was significantly enriched with increasing altitude and the duration of residency at high altitude,and the expression of the D-Ala metabolic pathway was higher in the high altitude group than in the middle altitude group and the initial high altitude group.(5)The trends of the device results are consistent with the sequencing results,which confirms the experimental accuracy of the in vitro device.Conclusion: This experiment successfully simulated the growth of oral microorganisms at different altitude oxygen concentrations using the designed-in vitro device,and the trend of bacterial growth in the device was the same as the one shown by 16 S r RNA high-throughput sequencing results.This study provides a theoretical basis for the phenomenon of increased prevalence of oral diseases in plateau-resident populations,and also provides a new in vitro experimental idea for the study of the oral microbiome and environmental factors.